Methods for Treating Heart Transplant Rejection

ABSTRACT

Current therapeutic approach to treating heart allotransplantation rejection focus on immunosuppression protocols that carry harmful side effects after chronic use, which include global immune depression to the patient. The present inventors have discovered alternative and synergistic protocols based on inhibiting NF-κB and NLRP3 inflammasome-dependent IL-1βrelease with nitrated NSAID derivatives.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/256,235 filed on Jan. 24, 2019, and claims the benefit of U.S.Provisional Application 62/621,228 filed Jan. 24, 2018.

BACKGROUND

Heart transplantation is currently the only definitive solution when aheart has total failure of its functions. In clinical practiceallografts of the heart are usually performed, necessarily carryingimmunosuppression protocols to avoid acute rejection. However, chronicrejection has not yet been successfully managed. In addition, the sideeffects associated with the chronic use of immunosupressants constitutea therapeutic problem due to their toxicity and the fact that theygenerate a global immunodepression to the patient. Currently,therapeutic strategies are sought to avoid their chronic use and highdoses of them. Until now, all therapies to avoid the rejection ofallotransplantation had as objective the manipulation of the adaptiveimmunity. Thus, there is a need to develop a novel therapeutic protocol,which includes a combination with classical immunosuppressants(administered in low doses and short duration) in order to obtainsynergistic and long-term effects for allograft survival.

SUMMARY

One embodiment of the described invention is a therapeutic approach totreat heart allotransplantation rejection based on the inflammasomeinhibition with a new anti-inflammatory nitroalkene.

One embodiment is a method of treating heart transplant rejectioncomprising administering to a subject in need thereof an effectiveamount of a compound of Formula I:

Another embodiment includes a method of treating heart transplantrejection comprising administering to a subject in need thereof aneffective amount of a compound of Formula I:

further comprising administering one or more secondary therapeuticagents.

Another embodiment includes a method of treating heart transplantrejection comprising administering to a subject in need thereof aneffective amount of a compound of Formula I:

further comprising administering one or more secondary therapeuticagents, wherein the one or more secondary therapeutic agents is selectedfrom the group consisting of calcineurin inhibitors, corticosteroids,cytotoxic immunosuppressants, immunosuppressant antibodies, sirolimusderivatives, other immunosuppressants, and any combination thereof.

One embodiment is method of treating heart transplant rejection in asubject comprising administering to said subject a pharmaceuticalcomposition comprised of an effective amount of a compound of Formula I:

or a pharmaceutically acceptable salt thereof, and a carrier.

Another embodiment includes a method of treating heart transplantrejection in a subject comprising administering to said subject apharmaceutical composition comprised of an effective amount of acompound of Formula I:

or a pharmaceutically acceptable salt thereof, and a carrier, whereinthe pharmaceutical composition further comprises one or more secondarytherapeutic agents.

Another embodiment includes a method of treating heart transplantrejection in a subject comprising administering to said subject apharmaceutical composition comprised of an effective amount of acompound of Formula I:

or a pharmaceutically acceptable salt thereof, and a carrier, whereinthe pharmaceutical composition further comprises one or more secondarytherapeutic agents selected from the group consisting of calcineurininhibitors, corticosteroids, cytotoxic immunosuppressants,immunosuppressant antibodies, sirolimus derivatives, otherimmunosuppressants, and any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the inhibitory effect of SANA on NF-κB and NLRP3inflamasome-dependent IL-1β release in the plasma of C57BL/6 mice.

FIG. 2 demonstrates the inhibitory effect of SANA on NF-κB and NLRP3inflamasome-dependent IL-1β release in the peritoneum of C57BL/6 mice.

FIG. 3 demonstrates prolonged survival for fully mismatched heterotopicheart allograft in rat models treated with SANA.

DESCRIPTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularprocesses, compositions, or methodologies described, as these may vary.It is also to be understood that the terminology used in the descriptionis for the purpose of describing the particular versions or embodimentsonly, and is not intended to limit the scope of the present inventionwhich will be limited only by the appended claims. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare now described. All publications mentioned herein are incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

It must also be noted that as used herein and in the appended claims,the singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference toa “cell” is a reference to one or more cells and equivalents thereofknown to those skilled in the art, and so forth.

As used herein, the term “about” means plus or minus 5% of the numericalvalue of the number with which it is being used. Therefore, about 50%means in the range of 45%-55%.

“Administering” when used in conjunction with a therapeutic means toadminister a therapeutic directly to a subject, whereby the agentpositively impacts the target. “Administering” a composition may beaccomplished by, for example, injection, oral administration, topicaladministration, or by these methods in combination with other knowntechniques. Such combination techniques include heating, radiation,ultrasound and the use of delivery agents. When a compound is providedin combination with one or more other active agents, “administration”and its variants are each understood to include concurrent andsequential provision of the compound or salt and other agents.

By “pharmaceutically acceptable” it is meant the carrier, diluent,adjuvant, or excipient must be compatible with the other ingredients ofthe formulation and not deleterious to the recipient thereof.

“Composition” as used herein is intended to encompass a productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationof the specified ingredients in the specified amounts. Such term inrelation to “pharmaceutical composition” is intended to encompass aproduct comprising the active ingredient(s), and the inert ingredient(s)that make up the carrier, as well as any product which results, directlyor indirectly, from combination, complexation or aggregation of any twoor more of the ingredients, or from dissociation of one or more of theingredients, or from other types of reactions or interactions of one ormore of the ingredients. Accordingly, the pharmaceutical compositions ofthe present invention encompass any composition made by admixing acompound of the present invention and a pharmaceutically acceptablecarrier.

As used herein, the term “agent,” “active agent,” “therapeutic agent,”or “therapeutic” means a compound or composition utilized to treat,combat, ameliorate, prevent or improve an unwanted condition or diseaseof a patient. Furthermore, the term “agent,” “active agent,”“therapeutic agent,” or “therapeutic” encompasses a combination of oneor more of the compounds of the present invention.

A “therapeutically effective amount” or “effective amount” of acomposition is a predetermined amount calculated to achieve the desiredeffect, i.e., to inhibit, block, or reverse the activation, migration,proliferation, alteration of cellular function, and to preserve thenormal function of cells. The activity contemplated by the methodsdescribed herein includes both medical therapeutic and/or prophylactictreatment, as appropriate, and the compositions of the invention may beused to provide improvement in any of the conditions described. It isalso contemplated that the compositions described herein may beadministered to healthy subjects or individuals not exhibiting symptomsbut who may be at risk of developing a particular disorder. The specificdose of a compound administered according to this invention to obtaintherapeutic and/or prophylactic effects will, of course, be determinedby the particular circumstances surrounding the case, including, forexample, the compound administered, the route of administration, and thecondition being treated. However, it will be understood that the chosendosage ranges are not intended to limit the scope of the invention inany way. A therapeutically effective amount of compound of thisinvention is typically an amount such that when it is administered in aphysiologically tolerable excipient composition, it is sufficient toachieve an effective systemic concentration or local concentration inthe tissue.

The terms “treat,” “treated,” or “treating” as used herein refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological condition, disorder, or disease, or to obtain beneficialor desired clinical results. For the purposes of this invention,beneficial or desired results include, but are not limited to,alleviation of symptoms; diminishment of the extent of the condition,disorder, or disease; stabilization (i.e., not worsening) of the stateof the condition, disorder, or disease; delay in onset or slowing of theprogression of the condition, disorder, or disease; amelioration of thecondition, disorder, or disease state; and remission (whether partial ortotal), whether detectable or undetectable, or enhancement orimprovement of the condition, disorder, or disease. Treatment includesprolonging survival as compared to expected survival if not receivingtreatment.

The term “subject,” as used herein, describes an organism, includingmammals, to which treatment with the compositions and compoundsaccording to the subject disclosure can be administered. Mammalianspecies that can benefit from the disclosed methods include, but are notlimited to, apes, chimpanzees, orangutans, humans, monkeys; and otheranimals such as dogs, cats, horses, cattle, pigs, sheep, goats,chickens, mice, rats, guinea pigs, and hamsters. Typically, the subjectis a human.

The term “tissue,” as used herein, describes an aggregate of cellstypically of a particular kind together with their intercellularsubstance that form one of the structural materials of a subject. Theterm “organ,” as used herein, describes a group of tissues that performa specific function. For example, heart is a type of organ embodiedherein.

Administration and Compositions

The compounds and pharmaceutically-acceptable salts thereof can beadministered by means that produces contact of the active agent with theagent's site of action. They can be administered by conventional meansavailable for use in conjunction with pharmaceuticals in a dosage rangeof 0.001 to 1000 mg/kg of mammal (e.g. human) body weight per day in asingle dose or in divided doses. One dosage range is 0.01 to 500 mg/kgbody weight per day orally in a single dose or in divided doses.Administration can be delivered as individual therapeutic agents or in acombination of therapeutic agents. They can be administered alone, buttypically are administered with a pharmaceutically acceptable excipientselected on the basis of the chosen route of administration and standardpharmaceutical practice.

Compounds can be administered by one or more ways. For example, thefollowing routes may be utilized: oral, parenteral (includingsubcutaneous injections, intravenous, intramuscular, intrasternalinjection or infusion techniques), inhalation, buccal, sublingual, orrectal, in the form of a unit dosage of a pharmaceutical compositioncontaining an effective amount of the compound and optionally incombination with one or more pharmaceutically-acceptable excipients suchas stabilizers, anti-oxidants, lubricants, bulking agents, fillers,carriers, adjuvants, vehicles, diluents and other readily knownexcipients in standard pharmaceutical practice.

Liquid preparations suitable for oral administration (e.g. suspensions,syrups, elixirs and other similar liquids) can employ media such aswater, glycols, oils, alcohols, and the like. Solid preparationssuitable for oral administration (e.g. powders, pills, capsules andtablets) can employ solid excipients such as starches, sugars, kaolin,lubricants, binders, disintegrating agents, antioxidants and the like.

Parenteral compositions typically employ sterile water as a carrier andoptionally other ingredients, such as solubility aids. Injectablesolutions can be prepared, for example, using a carrier comprising asaline solution, a glucose solution or a solution containing a mixtureof saline and glucose. Further guidance for methods suitable for use inpreparing pharmaceutical compositions is provided in Remington: TheScience and Practice of Pharmacy, 21^(st) edition (Lippincott Williams &Wilkins, 2006).

Therapeutic compounds can be administered orally in a dosage range ofabout 0.001 to 1000 mg/kg of mammal (e.g. human) body weight per day ina single dose or in divided doses. One dosage range is about 0.01 to 500mg/kg body weight per day orally in a single dose or in divided doses.For oral administration, the compositions can be provided in the form oftablets or capsules containing about 1.0 to 500 mg of the activeingredient, particularly about 1, 5, 10, 15, 20, 25, 50, 75, 100, 150,200, 250, 300, 400, 500, and 750 mg of the active ingredient for thesymptomatic adjustment of the dosage to the patient to be treated. Thespecific dose level and frequency of dosage for any particular patientmay be varied and will depend upon a variety of factors including theactivity of the specific compound employed, the metabolic stability andlength of action of that compound, the age, body weight, general health,sex, diet, mode and time of administration, rate of excretion, drugcombination, the severity of the particular condition, and the subjectundergoing therapy. In view of the factors affecting the specific doselevel and frequency it is contemplated that the dose frequency can rangefrom multiple doses daily to monthly dosages. The preferred dosefrequency ranges from twice a day to every two weeks. A more preferreddose frequency ranges from twice a day to weekly. A most preferred dosefrequency ranges from twice a day to twice a week.

In the methods of various embodiments, pharmaceutical compositionsincluding the active agent can be administered to a subject in an“effective amount.” An effective amount may be any amount that providesa beneficial effect to the patient, and in particular embodiments, theeffective amount is an amount that may: (1) prevent or reduce rejectionof heart tissue allografts and (2) prevent or reduce rejection of atransplanted heart.

Pharmaceutical formulations containing the compounds of the inventionand a suitable carrier can be in various forms including, but notlimited to, solids, solutions, powders, fluid emulsions, fluidsuspensions, semi-solids, and dry powders including an effective amountof an the active agent of the invention. It is also known in the artthat the active ingredients can be contained in such formulations withpharmaceutically acceptable diluents, fillers, disintegrants, binders,lubricants, surfactants, hydrophobic vehicles, water soluble vehicles,emulsifiers, buffers, humectants, moisturizers, solubilizers,antioxidants, preservatives and the like. The means and methods foradministration are known in the art and an artisan can refer to variouspharmacologic references for guidance. For example, ModernPharmaceutics, Banker & Rhodes, Marcel Dekker, Inc. (1979); and Goodman& Gilman's, The Pharmaceutical Basis of Therapeutics, 6th Edition,MacMillan Publishing Co., New York (1980) both of which are herebyincorporated by reference in their entireties can be consulted.

Other embodiments of the invention include the active agent prepared asdescribed above which are formulated as a solid dosage form for oraladministration including capsules, tablets, pills, powders, andgranules. In such embodiments, the active compound may be admixed withone or more inert diluent such as sucrose, lactose, or starch. Suchdosage forms may also comprise, as in normal practice, additionalsubstances other than inert diluents, e.g., lubricating agents such asmagnesium stearate. In the case of capsules, tablets, and pills, thedosage forms may also comprise buffering agents and can additionally beprepared with enteric coatings.

In another exemplary embodiment, an oily preparation of an active agentprepared as described above may be lyophilized to form a solid that maybe mixed with one or more pharmaceutically acceptable excipient, carrieror diluent to form a tablet, and in yet another embodiment, the activeagent may be crystallized to from a solid which may be combined with apharmaceutically acceptable excipient, carrier or diluent to form atablet.

The means and methods for tableting are known in the art and one ofordinary skill in the art can refer to various references for guidance.For example, Pharmaceutical Manufacturing Handbook: Production andProcesses, Shayne Cox Gad, John Wiley & Sons, Inc., Hoboken, New Jersey(2008), which is hereby incorporated by reference in its entirety can beconsulted.

Further embodiments which may be useful for oral administration of theactive agent include liquid dosage forms. In such embodiments, a liquiddosage may include a pharmaceutically acceptable emulsion, solution,suspension, syrup, and elixir containing inert diluents commonly used inthe art, such as water. Such compositions may also comprise adjuvants,such as wetting agents, emulsifying and suspending agents, andsweetening, flavoring, and perfuming agents. Thus, for example, thecompounds can be formulated with suitable polymeric or hydrophobicmaterials (for example, as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Other suitable diluents include, but are notlimited to those described below:

Vegetable oil: As used herein, the term “vegetable oil” refers to acompound, or mixture of compounds, formed from ethoxylation of vegetableoil, wherein at least one chain of polyethylene glycol is covalentlybound to the vegetable oil. In some embodiments, the fatty acids mayhave between about twelve carbons to about eighteen carbons. In someembodiments, the amount of ethoxylation can vary from about 2 to about200, about 5 to 100, about 10 to about 80, about 20 to about 60, orabout 12 to about 18 of ethylene glycol repeat units. The vegetable oilmay be hydrogenated or unhydrogenated. Suitable vegetable oils include,but are not limited to castor oil, hydrogenated castor oil, sesame oil,corn oil, peanut oil, olive oil, sunflower oil, safflower oil, soybeanoil, benzyl benzoate, sesame oil, cottonseed oil, and palm oil. Othersuitable vegetable oils include commercially available synthetic oilssuch as, but not limited to, Miglyol™ 810 and 812 (available fromDynamit Nobel Chemicals, Sweden) Neobee™ M5 (available from DrewChemical Corp.), Alofine™ (available from Jarchem Industries), theLubritab™ series (available from JRS Pharma), the Sterotex™ (availablefrom Abitec Corp.), Softisan™ 154 (available from Sasol), Croduret™(available from Croda), Fancol™ (available from the Fanning Corp.),Cutina™ HR (available from Cognis), Simulsol™ (available from CJPetrow), EmCon™ CO (available from Amisol Co.), Lipvol™ CO, SES, andHS-K (available from Lipo), and Sterotex™ HM (available from AbitecCorp.). Other suitable vegetable oils, including sesame, castor, corn,and cottonseed oils, include those listed in R. C. Rowe and P. J.Shesky, Handbook of Pharmaceutical Excipients, (2006), 5th ed., which isincorporated herein by reference in its entirety. Suitablepolyethoxylated vegetable oils, include but are not limited to,Cremaphor™ EL or RH series (available from BASF), Emulphor™ EL-719(available from Stepan products), and Emulphor™ EL-620P (available fromGAF).

Mineral oils: As used herein, the term “mineral oil” refers to bothunrefined and refined (light) mineral oil. Suitable mineral oilsinclude, but are not limited to, the Avatech™ grades (available fromAvatar Corp.), Drakeol™ grades (available from Penreco), Sirius™ grades(available from Shell), and the Citation™ grades (available from AvaterCorp.).

Castor oils: As used herein, the term “castor oil,” refers to a compoundformed from the ethoxylation of castor oil, wherein at least one chainof polyethylene glycol is covalently bound to the castor oil. The castoroil may be hydrogenated or unhydrogenated. Synonyms for polyethoxylatedcastor oil include, but are not limited to polyoxyl castor oil,hydrogenated polyoxyl castor oil, mcrogolglyceroli ricinoleas,macrogolglyceroli hydroxystearas, polyoxyl 35 castor oil, and polyoxyl40 hydrogenated castor oil. Suitable polyethoxylated castor oilsinclude, but are not limited to, the Nikkol™ HCO series (available fromNikko Chemicals Co. Ltd.), such as Nikkol HCO-30, HC-40, HC-50, andHC-60 (polyethylene glycol-30 hydrogenated castor oil, polyethyleneglycol-40 hydrogenated castor oil, polyethylene glycol-50 hydrogenatedcastor oil, and polyethylene glycol-60 hydrogenated castor oil,Emulphor™ EL-719 (castor oil 40 mole-ethoxylate, available from StepanProducts), the Cremophore™ series (available from BASF), which includesCremophore RH40, RH60, and EL35 (polyethylene glycol-40 hydrogenatedcastor oil, polyethylene glycol-60 hydrogenated castor oil, andpolyethylene glycol-35 hydrogenated castor oil, respectively), and theEmulgin® RO and HRE series (available from Cognis PharmaLine). Othersuitable polyoxyethylene castor oil derivatives include those listed inR. C. Rowe and P. J. Shesky, Handbook of Pharmaceutical Excipients,(2006), 5th ed., which is incorporated herein by reference in itsentirety.

Sterol: As used herein, the term “sterol” refers to a compound, ormixture of compounds, derived from the ethoxylation of sterol molecule.Suitable polyethoyxlated sterols include, but are not limited to, PEG-24cholesterol ether, Solulan™ C-24 (available from Amerchol); PEG-30cholestanol, Nikkol™ DHC (available from Nikko); Phytosterol, GENEROL™series (available from Henkel); PEG-25 phyto sterol, Nikkol™ BPSH-25(available from Nikko); PEG-5 soya sterol, Nikkol™ BPS-5 (available fromNikko); PEG-10 soya sterol, Nikkol™ BPS-10 (available from Nikko);PEG-20 soya sterol, Nikkol™ BPS-20 (available from Nikko); and PEG-30soya sterol, Nikkol™ BPS-30 (available from Nikko).

Polyethylene glycol: As used herein, the term “polyethylene glycol” or“PEG” refers to a polymer containing ethylene glycol monomer units offormula —O—CH₂—CH₂—. Suitable polyethylene glycols may have a freehydroxyl group at each end of the polymer molecule, or may have one ormore hydroxyl groups etherified with a lower alkyl, e.g., a methylgroup. Also suitable are derivatives of polyethylene glycols havingesterifiable carboxy groups. Polyethylene glycols useful in the presentinvention can be polymers of any chain length or molecular weight, andcan include branching. In some embodiments, the average molecular weightof the polyethylene glycol is from about 200 to about 9000. In someembodiments, the average molecular weight of the polyethylene glycol isfrom about 200 to about 5000. In some embodiments, the average molecularweight of the polyethylene glycol is from about 200 to about 900. Insome embodiments, the average molecular weight of the polyethyleneglycol is about 400. Suitable polyethylene glycols include, but are notlimited to polyethylene glycol-200, polyethylene glycol-300,polyethylene glycol-400, polyethylene glycol-600, and polyethyleneglycol-900. The number following the dash in the name refers to theaverage molecular weight of the polymer. In some embodiments, thepolyethylene glycol is polyethylene glycol-400. Suitable polyethyleneglycols include, but are not limited to the Carbowax™ and Carbowax™Sentry series (available from Dow), the Lipoxol™ series (available fromBrenntag), the Lutrol™ series (available from BASF), and the Pluriol™series (available from BASF).

Propylene glycol fatty acid ester: As used herein, the term “propyleneglycol fatty acid ester” refers to a monoether or diester, or mixturesthereof, formed between propylene glycol or polypropylene glycol and afatty acid. Fatty acids that are useful for deriving propylene glycolfatty alcohol ethers include, but are not limited to, those definedherein. In some embodiments, the monoester or diester is derived frompropylene glycol. In some embodiments, the monoester or diester hasabout 1 to about 200 oxypropylene units. In some embodiments, thepolypropylene glycol portion of the molecule has about 2 to about 100oxypropylene units. In some embodiments, the monoester or diester hasabout 4 to about 50 oxypropylene units. In some embodiments, themonoester or diester has about 4 to about 30 oxypropylene units.Suitable propylene glycol fatty acid esters include, but are not limitedto, propylene glycol laurates: Lauroglycol™ FCC and 90 (available fromGattefosse); propylene glycol caprylates: Capryol™ PGMC and 90(available from Gatefosse); and propylene glycol dicaprylocaprates:Labrafac™ PG (available from Gatefosse).

Stearoyl macrogol glyceride: Stearoyl macrogol glyceride refers to apolyglycolized glyceride synthesized predominately from stearic acid orfrom compounds derived predominately from stearic acid, although otherfatty acids or compounds derived from other fatty acids may be used inthe synthesis as well. Suitable stearoyl macrogol glycerides include,but are not limited to, Gelucire® 50/13 (available from Gattefossé).

In some embodiments, the diluent component comprises one or more ofmannitol, lactose, sucrose, maltodextrin, sorbitol, xylitol, powderedcellulose, microcrystalline cellulose, carboxymethylcellulose,carboxyethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, methylhydroxyethylcellulose, starch, sodiumstarch glycolate, pregelatinized starch, a calcium phosphate, a metalcarbonate, a metal oxide, or a metal aluminosilicate.

Exemplary excipients or carriers for use in solid and/or liquid dosageforms include, but are not limited to:

Sorbitol: Suitable sorbitols include, but are not limited to,PharmSorbidex E420 (available from Cargill), Liponic 70-NC and 76-NC(available from Lipo Chemical), Neosorb (available from Roquette),Partech SI (available from Merck), and Sorbogem (available from SPIPolyols).

Starch, sodium starch glycolate, and pregelatinized starch include, butare not limited to, those described in R. C. Rowe and P. J. Shesky,Handbook of Pharmaceutical Excipients, (2006), 5th ed., which isincorporated herein by reference in its entirety.

Disintegrant: The disintegrant may include one or more of croscarmellosesodium, carmellose calcium, crospovidone, alginic acid, sodium alginate,potassium alginate, calcium alginate, an ion exchange resin, aneffervescent system based on food acids and an alkaline carbonatecomponent, clay, talc, starch, pregelatinized starch, sodium starchglycolate, cellulose floc, carboxymethylcellulose,hydroxypropylcellulose, calcium silicate, a metal carbonate, sodiumbicarbonate, calcium citrate, or calcium phosphate.

Still further embodiments of the invention include the active agentadministered in combination with other active such as, for example,adjuvants, or other compatible drugs or compounds where such combinationis seen to be desirable or advantageous in achieving the desired effectsof the methods described herein.

Other embodiments of the present invention include a pharmaceuticalcomposition comprising an effective amount of the active agent and oneor more pharmaceutically acceptable excipient. Other embodiments includea pharmaceutical composition comprising an effective amount ofpharmaceutically-acceptable salts of the active agent. Other embodimentsinclude a pharmaceutical composition comprising an effective amount ofpharmaceutically-acceptable salts of active agent and apharmaceutically-acceptable excipient.

In yet other embodiments, the active agent may be administeredsimultaneously or separately with one or more secondary therapeuticagents. Secondary therapeutic agents may include but are not limited to:immunosuppressant agents such as calcineurin inhibitors (cyclosporin,tacrolimus), corticosteroids (methylprednisolone, dexamethasone,prednisolone), cytotoxic immunosuppressants (azathioprine, chlorambucil,cyclophosphamide, mercaptopurine, methotrexate), immunosuppressantantibodies (eg antithymocyte globulins, basiliximab, infliximab),sirolimus derivatives (everolimus, sirolimus), other immunosuppressants(mycophenolate), and any combination thereof.

The compound of Formula I and pharmaceutical compositions thereof asdescribed herein may be administered to subjects to treat tissueallograft rejection. In other embodiments, the compound of Formula I andpharmaceutical compositions thereof as described herein may beadministered to subjects to prevent or reduce rejection of atransplanted organ. In some embodiments the compound of Formula I andpharmaceutical compositions thereof as described herein may beadministered to subjects to prevent or reduce rejection of atransplanted heart. In some embodiments, the compound of Formula I andpharmaceutical compositions thereof as described herein may be used toprolong the survival of a transplanted heart.

EXAMPLES

The following examples contain detailed methods of preparing compoundsof Formula I. These detailed descriptions serve to exemplify the abovegeneral synthetic schemes which form part of the invention. Thesedetailed descriptions are presented for illustrative purposes only andare not intended as a restriction on the scope of the invention. Allparts are by weight and temperatures are in Degrees Celsius unlessotherwise indicated. All compounds showed NMR spectra consistent withtheir assigned structures.

Example 1 2-hydroxy-5-(2-nitroethenyl)benzoic acid (SANA)

To a solution of 5-formylsalicylic (1g, 6.02 mmol) in ethanol (16.5 mL),nitromethane (5.5 mL, 0.10 mmol) and ammonium acetate (1.39 g, 18.06mmol) were added. The reaction mixture is heated at 60° C. for 1 h,allowed to cool to room temperature and put in refrigerator for 15minutes. Formed orange precipitate was filtered off and dissolved inwater (ca. 250 mL). Solution was acidified with concentrated HC1 (ca. 10drops) until total precipitation. Formed yellow solid was filtered offand dried in vacuo. Yield: 1.18 g (93%).

1H NMR (acetone-d₆): δ=8.35 (d, J=2.3 Hz, 1H), 8.14 (d, J=13.7, 1H),8.06 (dd, J=8.7 2.3 Hz, 1H), 7.99 (d, J=13.7 Hz, 1H), 7.11 (d, J=8.7 Hz,1H). 13C NMR (acetone-d6): δ=171.09, 164.66, 137.94, 136.51, 135.94,133.21, 121.95, 118.51, 113.08

Biologic Activity

The following methods described are used in order to demonstratebiological activity and therapeutic use, and should not to be construedin any way as limiting the scope of the invention.

While not wishing to be bound by theory, NLRP3 inflammasome activationis a mechanism that mediates the rejection of allotransplantation and inone embodiment of the described invention, it is a possible therapeutictarget to treat allotransplantation of a heart. As shown in FIGS. 1 and2, SANA, in vivo, inhibits NF-KB and the NLRP3 inflammasome-dependentIL-1β release. C57BL/6 mice were treated with SANA or salicylic acid(SA) (100 mg/kg, IP) or the vehicle (DMSO) for 1 hour. Then wereinjected with LPS (10 mg/kg, IP) or PBS for 2 hours, with subsequentperitoneal washes and blood extractions. Peritoneal wash and plasma werestored to measure IL-10 by ELISA. FIG. 3 demonstrated SANA in a ratmodel, for a fully mismatched heterotopic heart allograft, prolonged theheart grafted survival respect to the control and its precursor drug.Rats were administrated with SANA or SA (100 mg/kg), SANA (50 mg/kg) orvehicle (Phosphate buffer) by oral gavage every day since day -1 untilday 15 post-transplantation. Heart heterotopic transplantation was doneby implantation of the donor heart (Lewis 1W rat) into the receptorabdomen (Lewis 1A rat). Rejection was diagnosed when the heart lostfunctionality (beats has stopped).

What is claimed is:
 1. A method of treating heart transplant rejectioncomprising administering to a subject in need thereof an effectiveamount of a compound of Formula I:


2. The method of claim 1, further comprising administering one or moresecondary therapeutic agents.
 3. The method of claim 2, wherein the oneor more secondary therapeutic agents is selected from the groupconsisting of calcineurin inhibitors, corticosteroids, cytotoxicimmunosuppressants, immunosuppressant antibodies, sirolimus derivatives,other immunosuppressants, and any combination thereof.
 4. A method oftreating heart transplant rejection in a subject comprisingadministering to said subject a pharmaceutical composition comprised ofan effective amount of a compound of Formula I:

or a pharmaceutically acceptable salt thereof, and a carrier.
 5. Themethod of claim 4, wherein the pharmaceutical composition furthercomprises one or more secondary therapeutic agents.
 6. The method ofclaim 5, wherein the one or more secondary therapeutic agents isselected from the group consisting of calcineurin inhibitors,corticosteroids, cytotoxic immunosuppressants, immunosuppressantantibodies, sirolimus derivatives, other immunosuppressants, and anycombination thereof.